The global increase in tuberculosis concomitant with the spread of HIV infection has led to renewed interested in Mycobacteria tuberculosis. Despite its long history as a pathogen, the definition of virulence determinants in this organism has been difficult. It is clear a crucial encounter between pathogen and host occurs when the bacteria is engulfed by alveolar macrophages. If the bacteria resist killing by the macrophage they can remain dormant in the host for decades re-emerging to cause disease when the host immune system is compromised by old age, HIV infection or chemotherapy. The goal of this research is to study the fact of pathogenic Mycobacteria within the host cell, to characterize cellular and bacterial components required for bacterial entry into and survival within host cells, and to identify bacterial components required for long term survival in the host. We are using Mycobacterium marinum and M. ulcerans, both of which cause persistent disease in humans, as models for studying M. tuberculosis. An extensive taxonomic analysis of both pathogenic and nonpathogenic Mycobacteria species shows M. marinum and M. ulcerans are more closely related to M. tuberculosis than any Mycobacteria species except for M. bovis. Using cultured cell lines we have shown that M. marinum replicates and persists within macrophages, epithelial cells and fibroblasts. Using confocal microscopy, we found that M. marinum, appears to traffick like M. tuberculosis in macrophage cell lines. We isolated an M. marinum mutant which enters but does not persist within macrophages. This mutant, when compared to the parental strain in guinea pig and frog models is seriously attenuated for virulence. The molecular nature of this attenuation is under scrutiny. As bacteria enter stationary phase they acquire a number of new phenotypes including resistance to nutrient deprivation, oxidative stress and the production of numerous proteins not made during exponential growth. In many species of bacteria the expression of genes encoding such traits is regulated by a "stationary phase" sigma factor. We have identified a putative alternative sigma factor present in M. tuberculosis and other pathogenic slow growing Mycobacteria species but absent from nonpathogenic fast growing species. This protein has significant similarity to the WhiG, a sigma factor in Streptomyces species necessary for hyphal formation. We are in the process of characterizing this gene and determining itsfunction.